Studies will be continued on the biochemical basis of cold- sensitivity with emphasis is on those mutations in pyrA and in genes encoding nucleotide kinases since these genes very frequently express mutations as a cold-sensitive phenotype. The pyrA mutations will be analyzed using in vitro reconstitution of holoenzymes from various combinations of the dissimilar subunits from wild type and various mutants. In addition the basis of the cold-sensitive, arginine minus phenotype will be analyzed by constructing double mutants between pyrA and various arginine genes to determine which intermediate of the arginine pathway inhibits pyrA function in the cold-sensitive mutants. The emphasis of these studies will be to assess the impact of subunit- subunit interaction on the expression of a cold-sensitive phenotype. In addition, experiments will be done to determine the reason that mutations in nucleoside kinases are so commonly expressed as a cold- sensitive phenotype. Previous studies have established that ribosomal mutants and mutations affecting regulatory proteins are frequently expressed as cold-sensitive. Nucleoside kinase mutants appear to represent a third major class of cold-sensitive mutants.